Application of pyrogallic acid in preparation of medicine for preventing and treating ulcerative colitis
Gallic acid addresses the problem of insufficient response from existing drugs by inhibiting the function of CD8+ T cells and Th17 cells, significantly improving the symptoms of ulcerative colitis and providing a new treatment approach.
Patent Information
- Application Number
- CN202411509848.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-28
- Publication Date
- 2025-01-10
AI Technical Summary
Existing drugs for treating ulcerative colitis are insufficiently responsive, and some patients are prone to relapse after discontinuing medication. There is a lack of effective new drugs that target T-cell regulation as a treatment approach.
Using pyrogallol as the active ingredient, it protects the intestinal barrier function by inhibiting the cytolytic function of CD8+ T cells and inhibiting the differentiation of Th17 cells, thereby reducing the production of IFN-γ and IL-17A.
Gallic acid significantly improves symptoms of ulcerative colitis, reduces intestinal inflammation, lengthens colon, and lowers disease activity index and histological score, providing a new treatment strategy.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedicine and pharmacy, and relates to the application of pyrogallic acid in preparing medicines for preventing and treating ulcerative colitis. Background Art
[0002] Ulcerative colitis is a type of inflammatory bowel disease, a type of intestinal disease mediated by abnormal immune activation. Abnormal immune response can lead to excessive infiltration of immune cells and release of proinflammatory cytokines, thereby causing persistent inflammation of the intestinal mucosa, and then destroying the intestinal barrier function. Patients may experience diarrhea with mucus, pus and blood in the stool, paroxysmal colon spasmodic pain, fever and other symptoms. In severe cases, it can cause intestinal perforation or rectal cancer. This type of disease is limited to the mucosa and submucosa of the large intestine, mostly located in the sigmoid colon and rectum, and can also extend to the descending colon or even the entire colon. Due to the persistent abnormal activation of the immune system, the course of the disease is long and prone to recurrence. At present, the commonly used clinical drugs for ulcerative colitis include aminosalicylic acid inhibitors, glucocorticoids, immunosuppressants, etc., but the above drugs have insufficient response, and some patients have the problem of recurrence after stopping the drug. Therefore, it is urgent to further find new drugs to promote the clinical treatment of ulcerative colitis.
[0003] Abnormal activation and dysfunction of T cells play a key role in the development of ulcerative colitis. Specific T cell subsets play different roles in different stages of inflammation, among which CD8 + T cells and Th17 cells are particularly important in the occurrence and progression of the disease. + As a type of effector T cell, T cells mainly mediate cytotoxic reactions by secreting cytokines such as interferon-γ (IFN-γ). + The number and activity of T cells increased significantly, and by attacking and destroying intestinal epithelial cells, they triggered severe tissue damage and inflammatory responses. On the other hand, Th17 cells are a type of helper T cells that produce proinflammatory cytokines. They promote the recruitment and activation of neutrophils by secreting IL-17, while enhancing the production of other proinflammatory factors, further expanding and maintaining the inflammatory response, leading to the destruction of intestinal barrier function and inducing the continuous recurrence of ulcerative colitis. Therefore, targeted T cell regulation may be a promising approach for the treatment of ulcerative colitis.
[0004] Pyrogallol is a naturally occurring trihydroxyphenol compound widely distributed in the plant kingdom with a molecular formula of C6H 3 (OH) 3, by inhibiting the nuclear factor-κB (NF-κB) signaling pathway, reducing the production of reactive oxygen species (ROS), and regulating immune cell function, it exerts antiviral, anti-inflammatory, antioxidant, and immunomodulatory pharmacological effects. Studies have shown that pyrogallol has therapeutic effects on disease models such as lupus erythematosus, Alzheimer's disease, and psoriasis, but whether pyrogallol has a therapeutic effect on ulcerative colitis has not yet been clarified. Summary of the invention
[0005] The purpose of the present invention is to provide the use of pyrogallic acid in the preparation of drugs for preventing and treating ulcerative colitis. Pyrogallic acid has a CAS number of 87-66-1 and a molecular formula of C6H 3 (OH) 3 , molecular weight 126.11, molecular structure formula is as follows: Pyrogallol
[0006] The ulcerative colitis refers to an intestinal disease caused by abnormal immune activation.
[0007] The drug is a drug for improving the disease activity index (DAI) and colon length of ulcerative colitis.
[0008] The drug can inhibit CD8 + The cytolytic function of T cells can be used to reduce intestinal inflammation, and the inhibition of this function is specifically manifested in the reduction of IFN-γ secretion.
[0009] The drug may reduce the production of pro-inflammatory cytokines by inhibiting the differentiation of Th17 cells.
[0010] The drug is administered orally once a day, with a dosage of 75 mg / kg to 150 mg / kg
[0011] The present invention also provides a pharmaceutical composition composed of pyrogallic acid and pharmaceutically acceptable adjuvants and containing excipients, and the corresponding pharmaceutical dosage form is preferably one or more of capsules, tablets, oral suspensions, injections, pills, and lyophilized powder injections.
[0012] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention first discovered that pyrogallic acid has a good improvement and therapeutic effect on ulcerative colitis. Pyrogallic acid reduces CD8 + The two mechanisms of IFN-γ secreted by T cells and inhibition of Th17 cell differentiation to reduce the production of proinflammatory cytokine IL-17 work synergistically to reduce inflammatory responses in the intestine and protect intestinal barrier function, providing a new treatment strategy for ulcerative colitis with good prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The present invention found that pyrogallic acid can significantly improve the symptoms of female ulcerative colitis model mice. The results showed that pyrogallic acid can reduce body weight changes, reduce disease activity index DAI and colon histology scores, and prolong colon length.
[0014] Figure 2 The present invention found that pyrogallic acid can significantly improve the symptoms of male ulcerative colitis model mice. The results showed that pyrogallic acid can reduce body weight changes, reduce disease activity index DAI and colon histology scores, and prolong colon length.
[0015] Figure 3 The present invention found through cell experiments that pyrogallic acid can inhibit CD8 + The results showed that pyrogallic acid administration significantly reduced the expression of IFN-γ and the production of IL-17A. DETAILED DESCRIPTION
[0016] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents, instruments and methods used in the following examples are usually obtained through commercial channels under conventional conditions or according to the conditions recommended by the manufacturer without special instructions. Graph Pad Prism 9.0.1 software was used for statistical analysis, and the data were expressed as MEAN±SD. t-tests were used to analyze the differences between the two groups, and One-way ANOVA was used for variance analysis of multiple groups of data. *, P<0.05; **, P<0.01; ***, P<0.001.
[0017] Embodiment 1: Considering that there are differences in the DSS-induced colitis model between different sexes, mainly in that male mice are usually more sensitive to DSS-induced inflammation and show more severe intestinal damage and inflammatory response, while female mice may show milder symptoms under the protective effect of sex hormones. We established acute ulcerative colitis models in male and female mice and explored the therapeutic effect of pyrogallic acid on them.
[0018] The specific method is as follows: 6-8 week old male wild-type C57BL / 6 mice were divided into control group, model group and low-dose and high-dose pyrogallic acid groups. The mice in the control group drank normal water, while the mice in the model group and the treatment group drank water containing 4% DSS to induce colon inflammation. The mice in the treatment group were treated with DSS and given 75 mg / kg and 150 mg / kg pyrogallic acid ( Figure 1A). During the experiment, the weight changes of mice were monitored every day, and the mice were scored according to the disease activity scoring rules based on the three indicators of weight loss percentage, stool viscosity and fecal occult blood. Finally, the sum of the three scores was taken as the final DAI score, which was used to determine the severity of colitis in the experimental mice.
[0019] At the peak of the disease (5 days after DSS treatment), the mice were killed, the complete colon was taken, and the colon length was measured and recorded. The colon tissue fixed with 4% paraformaldehyde was taken, dehydrated, paraffin-embedded, and sectioned. The tissue morphology was observed by hematoxylin-eosin (H&E) staining, and judged from five indicators including inflammatory infiltration, goblet cell loss, and crypt density. Finally, the sum of the five scores was taken as the final histopathological score.
[0020] Figure 1 The results showed that compared with the model group, the weight of the mice in the pyrogallic acid group was reduced, and the DAI score was significantly reduced ( Figure 1 B). The colon length of mice in the model group was significantly shortened, and obvious inflammatory cell infiltration was observed in the submucosal layer of the colon, crypt structure damage, a large number of goblet cells were lost, and the pathological histological score was significantly increased. Compared with the model group, the colon length of mice in the pyrogallic acid administration group was significantly increased ( Figure 1 C), the infiltration area of inflammatory cells decreased, the edema of the submucosal layer was relieved, the crypt structure was intact, and the pathological histological score was significantly reduced ( Figure 1 D). The above results show that pyrogallic acid can effectively alleviate the symptoms of ulcerative colitis model in male mice.
[0021] Female wild-type C57BL / 6 mice aged 6-8 weeks were divided into a control group, a model group, and a pyrogallic acid administration group. The mice in the control group drank normal water, while the mice in the model group and the administration group drank water containing 4% DSS to induce colon inflammation. The mice in the administration group were treated with DSS and given pyrogallic acid (100 mg / kg) orally every day. Figure 2 A). During the experiment, the weight changes of mice were monitored every day, and the mice were scored according to the disease activity scoring rules based on the three indicators of weight loss percentage, stool viscosity and fecal occult blood. Finally, the sum of the three scores was taken as the final DAI score, which was used to determine the severity of colitis in the experimental mice.
[0022] At the peak of the disease (6 days after DSS treatment), the mice were killed, the complete colon was taken, and the colon length was measured and recorded. The colon tissue fixed with 4% paraformaldehyde was taken, dehydrated, paraffin-embedded, and sectioned. The tissue morphology was observed by hematoxylin-eosin (H&E) staining, and judged from five indicators including inflammatory infiltration, goblet cell loss, and crypt density. Finally, the sum of the five scores was taken as the final histopathological score.
[0023] Figure 2 The data showed that pyrogallic acid administration could significantly slow down the weight loss of mice after modeling, and the DAI score decreased from 7.83±1.55 to 5.92±1.43 ( Figure 2 B). Compared with the control group, the colon length of the model group mice was significantly shortened, accompanied by severe inflammatory cell infiltration in the submucosal layer of the colon, uneven mucosal surface, distorted crypt structure, and the pathological histological score increased from 2.33±0.58 to 9.00±2.00. In contrast, the colon length of the mice in the pyrogallic acid treatment group increased from 6.07±0.58cm to 7.27±0.51cm, which was a significant difference ( Figure 2 C), the infiltration of inflammatory cells in the submucosal layer decreased, the mucosal surface returned to a smooth state, the crypt structure was normal, and the histopathological score decreased to 7.67±1.15 ( Figure 2 D) These results suggest that pyrogallic acid has a significant alleviating effect on ulcerative colitis in female mice.
[0024] Embodiment 2: Thawed human PBMCs were cultured at 1x 10 6 The cells were seeded into 24-well plates containing 500 μL of 1640+10% Gi medium overnight, stimulated with 4% Brefeldin A for 1 h, and then treated with 20, 40, and 80 μM pyrogallic acid, respectively. Cell samples were collected after stimulation with 25 μL anti-CD3 / 28 Dynabeads for 18 h, and flow cytometry was used to co-stain CD3 and CD8 to mark the membrane surface maker. After staining for 30 min, the membrane was broken and stained for IFN-γ in order to investigate CD8 + The secretion of IFN-γ by T cells. Figure 3 A The results showed that pyrogallic acid could significantly reduce the expression of IFN-γ, indicating that pyrogallic acid could inhibit CD8 + T cell lytic function.
[0025] Embodiment 3: Enrichment of CD4 from human PBMCs by biotin-magnetic bead sorting +T cells were pretreated in 48-well plates with anti-CD3 (5 μg / ml), anti-CD28 (5 μg / ml), anti-IFNγ (10 μg / ml), and anti-IL-4 (10 μg / ml) antibodies. After 2 to 4 hours, the enriched CD4 + T cells were expressed at 3 x 10 5 / well was inoculated into a pre-coated 48-well plate containing 500 μL of 1640 + 10% Gi medium, and stimulated with IL-6 (20 ng / ml), IL-23 (25 ng / ml), IL-1β (12.5 ng / ml), IL-21 (25 ng / ml), and TGF-β (10 ng / ml), and then treated with 10 μM pyrogallic acid, cultured for 5 days to induce their differentiation into Th17 cells, and the cell supernatant was collected and the secretion of IL-17A was detected by ELISA kit. Figure 3 B The results showed that pyrogallic acid could significantly reduce the expression of IL-17A, indicating that pyrogallic acid could inhibit Th17 cell differentiation.
[0026] The above results indicate that pyrogallic acid can synergistically inhibit CD8 + The cytolytic function of T and the differentiation of Th17 cells can improve ulcerative colitis.
Claims
1. Use of pyrogallic acid in the preparation of a drug for preventing and treating ulcerative colitis, characterized in that: The molecular structure of pyrogallic acid is as follows:
2. The use according to claim 1, characterized in that: The ulcerative colitis refers to an intestinal disease caused by abnormal immune activation.
3. The use according to claim 1, characterized in that: The medicine is a medicine for improving disease activity index and colon length.
4. The use according to claim 1, characterized in that: The drug inhibits CD8 + The cytolytic function of T cells is used to reduce intestinal inflammation, and the inhibition of this function is specifically manifested in the reduction of interferon-γ secretion.
5. The use according to claim 1, characterized in that: The drug reduces the production of pro-inflammatory cytokines by inhibiting the differentiation of Th17 cells.
6. The use according to claim 1, characterized in that: The drug is administered orally once a day, with a dosage of 75 mg / kg to 150 mg / kg.
7. The use according to claim 1, characterized in that: The drug is made of pharmaceutically acceptable excipients, and the dosage form of the drug is one or more of capsules, tablets, oral suspensions, injections, pills, and freeze-dried powder injections.